Venezuelan equine encephalitis virus (VEEV) causes epidemics of neurological disease with high mortality, and can be spread by aerosol transmission. Thus, it has been categorized as an NIAID Class B priority pathogen. Currently, no licensed anti-viral agents exist for treatment of VEEV or other alphaviruses. Development of anti-viral agents for pre-exposure and post-exposure prophylaxis in the case of a large scale bioterrorist attack is important, given the high fatality rate of the disease caused by this alphavirus. The window of treatment, efficacy, and delivery issues cannot be addressed until such drugs are developed. Here we propose to validate a cell-free system for VEEV capsid assembly that can be developed in the future as a high throughput screen for identification of assembly inhibitors. Recent studies with other viruses have validated Inhibitors of capsid assembly potentially efficacious anti-viral drugs. In this application, we provide preliminary data indicating highly efficient assembly of VEEV capsids in the cell-free system. We propose to validate our findings by: 1) using transmission electron microscopy and other assays to confirm that the VEEV capsids made in the cell-free system closely resemble authentic capsids; 2) using mutational analysis to define domains of VEEV capsid protein (CP) needed for assembly; and 3) using RNA encapsidation studies to define the VEEV packaging signal. Our group has extensive experience in developing cell-free capsid assembly systems. For the proposed studies we will collaborate with investigators at USAMRIID who developed the VEEV replicon system. In addition, we are partnered with a small company that has a compound library and will use our assay for high throughput screening, as soon as it is validated by the studies proposed here. The cell-free VEE assembly system offer a safe and low cost approach to understanding the mechanism of VEE capsid formation and to screen for agents active against alphaviruses that have potential as bioterrorism agents.